Measurement of antimicrobial activity under reducing conditions in a modified radial diffusion assay

Abstract

This protocol describes the measurement of antimicrobial activity under reducing conditions against anaerobic bacteria of the human normal flora. The protocol is modified from the original description by Robert Lehrer et al. (1991), which is well established for the analysis of antimicrobial peptides with aerobic bacteria. To perform analysis for facultative and strict anaerobes we modified the assay by the addition of the reducing agent DTT to assay medium and performed incubation steps in an anaerobic jar. It was successfully used with analysis of Bifidobacteria, Lactobacilli, Bacteroides and Escherichia coli and might be adapted to other anaerobic bacteria. Several antimicrobial peptides have been tested with that system: while most of them showed less antimicrobial activity under reducing conditions, human beta defensin-1 was only active under these conditions against some of the mentioned strains.

Introduction

This protocol describes the measurement of antimicrobial activity under reducing conditions against anaerobic bacteria of the human normal flora. The protocol is modified from the original description by Robert Lehrer et al. (1), which is well established for the analysis of antimicrobial peptides with aerobic bacteria. To perform analysis for facultative and strict anaerobes we modified the assay by the addition of the reducing agent DTT to assay medium and performed incubation steps in an anaerobic jar. It was successfully used with analysis of Bifidobacteria, Lactobacilli, Bacteroides and Escherichia coli (2) and might be adapted to other anaerobic bacteria. Several antimicrobial peptides have been tested with that system: while most of them showed less antimicrobial activity under reducing conditions, human beta defensin-1 was only active under these conditions against some of the mentioned strains.

add ddH2O to a final volume of 50 ml (Note: If melting of the gel in the microwave causes significant loss of liquid it is possible to add more ddH2O in order to keep a remainder of 50 ml after microwaving.)

adjust pH to 7.4 with diluted HCl (1:100 or 1:10) (Note: depending on the number of gels you prepare only few drops might be required.)

autoclave in 100 ml bottle for 20 min at 121°C, store for short term at room temperature

Overlay „high nutrition“ gel (5 gels)

add 0.5 g EEO-agarose into 100 ml bottle

add 3 g TSB powder

add 5 ml of Phosphate buffer 100 mM, pH 7.4

add ddH2O to a final volume of 50 ml (Note: If melting of the gel in the microwave causes significant loss of liquid it is possible to add more ddH2O in order to keep a remainder of 50 ml after microwaving.)

autoclave in 100 ml bottle for 20 min at 121°C, store for short term at room temperature

B. Bacterial Preparation

Prepare anaerobic bacteria from kryo-culture

Incubate on Columbia Blood-Agar plates in an anaerobic jar for about 48 hours

Transfer bacteria into 50 ml falcon containing 10 ml 1x TSB

Place Falcon without lid into anaerobic jar, incubate for about 16 hours at 37°C without shaking. (Note: Since different bacterial strains behave differently optimal culture conditions should be optimized. Some bacteria might require shorter incubation times while others might require more time to produce optimal numbers of cells required for the assay.)

5 – 20 µl DTT can now be added into liquid gel from the 1 M stock solution. (Note: It is important to use freshly prepared DTT since repeated freezing and thawing impairs its function. Furthermore, some bacterial strains tolerate higher amounts of DTT while others do not. We obtained best results between concentrations of 0.5 and 2 mM (final) whereas 10 mM were mostly lethal. Depending on bacteria used different amounts of DTT might lead to best results.

10 – 20 µl resazurine can be added into liquid gel (final concentration 1 – 2 µg/ ml). (Note: The addition of this redox-indicator is a good opportunity to control reducing conditions in the underlay-gel. A “classical gel” should stain light blue while a reduced gel should look pink.)

Adjust to OD620nm = 0.1 and use 150 – 300 µl for assay (Note: The optimal volume of bacterial culture should be determined after the first experiments depending on the bacterial lawn obtained after the over-night incubation of the assay plates. In our hands it was optimal to use 150 µl for Bifidobacteria and 300 µl for Lactobacilli. For fast-growing bacteria like E. coli the user should refer to the classical protocol of Lehrer et al. (1))

Add bacterial suspension into 10 ml of warm, prepared underlay gel, mix gently. (Note: It is important that the gel is not too hot, which would have an effect on or kill bacterial cells, and not too cold since the gel would solidify. A temperature range between 42°C and 46°C should be fine for mixing with bacterial cells.)

Quickly pour the gel containing bacteria into square-petri-dishes, placed on an even surface, and let it solidify for about 30 minutes at room temperature. (Note: By letting a small opening between plate and its lid moisture level may be reduced, which improves punching of the wells later on. For strict anaerobic bacteria solidification time should be minimized).

Punch 6×6 wells into the gel and discard gel pieces with a sterile pipette tip or Pasteur-pipette connected to a vacuum pump

Fill up to 5 µl of proteins, dissolved in H2O or 0.01% acetic acid, into the wells. Include negative control (H2O or 0.01% acetic acid) and positive control (for example lysozyme, depending on bacteria tested)

Let protein solutions diffuse into the gel (short storage at 37°C or room temperature) and place plates into anaerobic jar for 3 hours.

Place plate upside-down in anaerobic incubator until bacterial lawn is clearly visible (16 to 48 hours)

Troubleshooting

1) No bacterial lawn after incubation.

– Bacteria added to liquid gel might not have been viable. Plate some remaining microliters of the over-night culture on Columbia Blood Agar plates and incubate anaerobically as a control
– Liquid gel might have been too hot. Reduce maintaining temperature or wait a little bit longer before mixing with bacteria
– For strict anaerobic bacteria oxygen exposure might have been to long
– DTT concentration might have been too high

2) Bacterial lawn is too thick, no inhibition zones are visible

– Use less volume of bacterial culture for underlay gel
– Reduce incubation time for the primary overnight culture

3) No inhibition zones are visible

– Inappropriate positive control (for example lysozyme does not work for Bacteroides vulgatus)
– Proteins/ Peptides are not functional under reducing conditions
– Too much moisture in the underlay gel has pressed protein solution out of the wells